[Performance of vaginal self-sampling high-risk HPV genotyping as primary and combining cytology or viral load as secondary in cervical cancer screening].

Objective: To evaluate the efficacy of high-risk HPV (HR-HPV) genotyping with vaginal self-sampling in primary screening and combining cytology or viral load for HR-HPV positive as secondary screening strategies. Methods: The data referring to HR-HPV genotyping of self-collected sample with mass array matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), HR-HPV viral load of physician-collected sample with hybrid capture Ⅱ (HC-Ⅱ), liquid-based cytology and histology of 8 556 women were from Shenzhen cervical cancer screening trial Ⅱ (SHENCCAST-Ⅱ) conducted between April 2009 and April 2010. The data were reanalyzed to determine the sensitivity and specificity to cervical intraepithelial neoplasia (CIN) of grade 2 or worse (CIN Ⅱ+), CIN of grade 3 or worse (CIN Ⅲ+) when HR-HPV genotyping combining with colposcopy as primary screening strategy based on varied HR-HPV subtype (strategy 1, including 5 sub-strategies: 1a: HPV 16/18 positive; 1b: HPV 16/18/58 positive; 1c: HPV 16/18/58/31/33 positive; 1d: HPV 16/18/58/31/33/52 positive; 1e: any HR-HPV positive). The data were also compared to determine the efficacy of cytology (strategy 2, including 5 sub-strategies: 2a, 2b, 2c, 2d, 2e) or HR-HPV viral load (strategy 3, including 4 sub-strategies: 3a, 3b, 3c, 3d) of physician-collected sample as a triage with HR-HPV genotyping for self-sampling HR-HPV positives. Results: (1) The HR-HPV positive rate was 13.77% (1 178/8 556) in the self-collected samples of 8 556 pregnant women. Of them,the prevalences of HPV 16/18, HPV 16/18/58, HPV 16/18/58/31/33 and HPV 16/18/58/31/33/52 were 3.16% (270/8 556), 5.14% (440/8 556), 6.66% (570/8 556) and 9.81% (839/8 556), respectively. The HR-HPV viral load ≥10 relative light units/control (RLU/CO) was 8.87%(759/ 8 556), while cytological results ≥atypical squamous cell of undetermined signification (ASCUS) were 12.05% (1 031/8 556). (2) The strategy 1e had the highest sensitivities for CIN Ⅱ+, CIN Ⅲ+ which were 92.70% and 94.33%,respectively,among 14 sub-strategies,while the lowest specificity and positive predictive value (PPV). Meanwhile,the required colposcopy referral rates were much higher than other 13 sub-strategies (13.77%). The other 4 sub-strategies of strategy 1 (1a, 1b, 1c, 1d), strategy 1a had the highest specificities for CIN Ⅱ+ and CIN Ⅲ+ (97.92%, 97.69%, respectively), while 1d had the highest sensitivities for CIN Ⅱ+ and CIN Ⅲ+ (88.41%, 92.20%, respectively). (3) Both strategies of referring self-sampling HPV 16/18 positives for immediate colposcopy followed by triage physician-collected sample cytology (≥ASCUS) or viral load (≥10 RLU/CO) for non-HPV 16/18 positives had significantly higher sensitivity and specificity for CIN Ⅱ, CIN Ⅲ+, as well as lower referral rates (strategy 2a and 3a). Additionally, based on these two secondary screening strategies, cumulatively using the other four HR-HPV (HPV 58, 31, 33 and 52) positives as triage for immediate colposcopy showed an enhanced sensitivity. Conclusions: Primary HR-HPV cervical cancer screening strategy based on self-sampling with triage of cytology (≥ASCUS) or viral load (≥10 RUL/CO) provides a good balance among sensitivity, specificity for CIN Ⅱ+ and CIN Ⅲ+ and the number of tests required, referral rates. The efficacy of HR-HPV genotyping combining cytology or viral load secondary screening strategies will have a spiral escalation when HPV 58, 31, 33, 52 are included.

[Clinical value of p16INK4a immunocytochemistry in cervical cancer screening].

Objective: To evaluate the value of p16INK4a detected by p16INK4a immunostaining as a new generation of cervical cytology for primary screening and secondary screening in population-based cervical cancer screening, and in improving cytological diagnosis. Methods: Between 2016 and 2018, 5 747 non-pregnant women aged 25-65 years with sexual history were recruited and underwent cervical cancer screening via high-risk (HR)-HPV/liquid-based cytological test (LCT) test in Shenzhen and surrounding areas. All slides were immuno-stained using p16INK4a technology, among them, 902 cases were offered p16INK4a detection during primary screening, and the remaining 4 845 cases were called-back by the virtue of abnormal HR-HPV and LCT results for p16INK4a staining. Participants with complete LCT examination, HR-HPV test, p16INK4a staining and histopathological examination results were included in this study. The performance of p16INK4a in primary and secondary screening, and in assisting cytology to detect high grade squamous intraepithelial lesion [HSIL, including cervical intraepithelial neoplasia (CIN) Ⅱ or Ⅲ] or worse [HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+] were analyzed. Results: (1) One-thousand and ninety-seven cases with complete data of p16INK4a and histology were included. Pathological diagnosis: 995 cases of normal cervix, 37 cases of low grade squamous intraepithelial lesion (LSIL), 64 cases of HSIL and one case of cervical cancer were found. Among them, 65 cases of HSIL (CIN Ⅱ)+ and 34 cases of HSIL (CIN Ⅲ)+ were detected. The positive rate of p16INK4a in HSIL (CIN Ⅱ)+ was higher than that in CINⅠ or normal pathology (89.2% vs 10.2%; P<0.01). (2) p16INK4a as primary screening for HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ was equally sensitive to primary HR-HPV screening (89.2% vs 95.4%, 94.1% vs 94.1%; P>0.05), but more specific than HR-HPV screening (89.8% vs 82.5%, 87.7% vs 80.2%; P<0.05). p16INK4a was equally sensitive and similarly specific to cytology (≥LSIL; P>0.05). (3) The specificity of LCT adjunctive p16INK4a for detecting HSIL (CIN Ⅱ)+ or HSIL (CIN Ⅲ)+ were higher than that of LCT alone or adjunctive HR-HPV (P<0.01), while the sensitivity were similar (P>0.05). (4) p16INK4a staining as secondary screening: p16INK4a was significantly more specific (94.1% vs 89.7%, 91.9% vs 87.4%; P<0.01) and comparably sensitive (84.6% vs 90.8%, 88.2% vs 91.2%; P>0.05) to cytology for triaging primary HR-HPV screening. HPV 16/18 to colposcopy and triage other HR-HPV with p16INK4a was equally sensitive (88.2% vs 94.1%; P=0.500) and more specific (88.3% vs 83.0%; P<0.01) than HPV 16/18 to colposcopy and triage other HR-HPV with LCT≥ atypical squamous cells of undetermined significance (ASCUS), and the referral rate decreased (14.0% vs 19.4%; P=0.005). Conclusions: For primary screening, p16INK4a is equally specific to cytology and equally sensitive to HR-HPV screening. p16INK4a alone could be an efficient triage after primary HR-HPV screening. In addition, p16INK4a immunostaining could be used as an ancillary tool to cervical cytological diagnosis, and improves its accuracy in cervical cancer screening.

[Evaluation of the effectiveness of BMRT-HPV for cervical cancer screening].

Objective: Evaluation of the clinical value of the BioPerfectus multiplex real time (BMRT)-HPV for cervical cancer screening. Methods: Physician-collected specimens of 1 495 women who were positive of Cobas 4800 HPV (Cobas-HPV), HPV genotyping based on SEQ uencing (SEQ-HPV), and (or) cytology ≥low grade squamous intraepithelial lesion (LSIL) in the primary screening of Chinese Multiple-center Screening Trial (CHIMUST), and 2 990 women selected from those who were negative of primary screening in the same project through nested control randomization with age-matching were tested for BMRT-HPV, which reported type-specific viral loads/10 000 cells in each specimen. With comparing to Cobas-HPV results and taking cervical histopathological diagnosis as the endpoint, the concordance of high-risk (HR)-HPV subtypes among the three assays was explored ,and the sensitivity and specificity of BMRT-HPV for cervical cancer screening were evaluated. Results: (1) The overall agreenment of HR-HPV subtypes between BMRT-HPV and Cobas-HPV, or SEQ-HPV test sample was 94.8%, 94.4%, with Kappa values 0.827, 0.814. (2) The sensitivity and specificity for cervical intraepithelial neoplasia (CIN) Ⅱ+ of BMRT-HPV, Cobas-HPV and SEQ-HPV were 92.62%, 94.26%, 93.44% and 84.67%, 83.25%, 82.76%, respectively. There were no significant difference in sensitivity among the three HPV assays (all P>0.05), but the specificity of BMRT-HPV for CIN Ⅱ+ was higher than those of Cobas-HPV and SEQ-HPV (P<0.01). The sensitivity for CIN Ⅲ+ of three HPV assays were all 100.00%, and the specificity for CIN Ⅲ+ of BMRT-HPV was higher than those of Cobas-HPV and SEQ-HPV (83.40% vs 81.95%, 83.40% vs 81.50%; P<0.01). The number of pathological examinations of colposcopy for cervical biopsy detected in 1 case of CIN Ⅱ+ or CIN Ⅲ+ in BMRT-HPV was less than those in Cobas-HPV and SEQ-HPV (P<0.01). When using HPV 16/18 + cytology ≥atypical squamous cell of undetermined signification (ASCUS) to triage HPV positive women among three assays, there was no different in the sensitivities of detecting CIN Ⅱ+ and CIN Ⅲ+ (P>0.05). The specificity BMRT-HPV was slightly higher than those in Cobas-HPV or SEQ-HPV (all P<0.05), and the colposcopy referral rate was lower than those in Cobas-HPV and SEQ-HPV (all P<0.05). Conclusions: BMRT-HPV is as sensitive as Cobas-HPV or SEQ-HPV for primary cervical cancer screening, and has higher specificity. Therefore it could be used as a primary screening method for cervical cancer, which is worthy of clinical application.

[The value of p16(INK4a) cytology for early diagnosis of cervical cancer].

Objective: To investigate the use of p16(INK4a) immuno-stained cytology as the primary screening for cervical cancer prevention. Methods: From March to August 2018, 902 women from Shenzhen and surrounding area were recruited for cervical cancer screening with ThinPrep Cytologic Test (TCT), cobas4800 HPV test, and p16(INK4a) co-test. Colpo/biopsies were performed using the point of interest biopsy protocol of directed and random cervical biopsies plus endocervical curettage for all women, any of whose tests was positive. Two senior cytopathologists interpreted TCT and p16(INK4a) test. The performance of p16(INK4a) for early detection of CIN2+ and inter-observer reproducibility of the interpretation of p16(INK4a) were evaluated. Results: The positive rates of HPV test, p16(INK4a) co-test and TCT diagnosed as LSIL/AGC or higher grade were 8.1% (73/902), 6.8% (61/902) and 4.7% (42/902), respectively. Colposcopy referring rate was 79.6% (109/137), among which 10 cases were diagnosed as CIN2+ (5 cases of CIN2 and 5 cases of CIN3). The sensitivity and specificity for CIN2+ of p16(INK4a) test, TCT (LSIL/AGC or higher grade) and HPV test were 90.0%, 80.0%, 100.0% and 90.9%, 91.9%, 82.5%, respectively. Compared to TCT and HPV test, there was no significant difference in sensitivity and specificity between p16(INK4a) and TCT/HPV test (P>0.05). The Kappa value of the 2 cytopathologists in interpreting p16(INK4a) and TCT was 0.944 and 0.425, respectively (P<0.05). Conclusions: p16(INK4a) for cervical cancer screening is equally sensitive to HPV test and specific to TCT while subjective difference of cytopathologists' interpretation of p16(INK4a) is small. Therefore, p16(INK4a) can be used as a new cervical cancer screen method for its better diagnostic performance.

[Relationship between cervical lesions and the type-specific viral load of high risk HPV reflected by the Ct value of Cobas 4800 HPV system].

Objective: To explore the relationship between cervical lesions and high risk HPV (HR-HPV) viral load reflected by the cycle threshold (Ct) values of Cobas 4800 HPV (Cobas 4800) system. Methods: From August 2016 to September 2017, 7 000 women from Shenzhen, were recruited for cervical cancer screening with Cobas 4800 system and cytology co-test. Colposcope biopsies were performed on women who were positive of HPV 16, 18, and positive of HPV types other than 16,18 with cytology [≥ atypical squamous cell of undetermined signification (ASCUS)], or HPV negative but abnormal of cytology [≥ low grade squamous intraepithelial lesion (LSIL)]. The Ct values of HPV 16, 18 and all combined other types coming from Cobas 4800 system were used as an indicator of viral load to analyze the relationship between type-specific HPV load and the cervical lesions. Results: (1) Among the 7 000 screening women, 370 cases were positive for cervical cancer screening, 325 of them underwent colposcope biopsies, and coloposcopy referred rate was 87.8% (325/370). Among 325 women undergoing cervical biopsy, pathological diagnosis was 119 cases of normal cervical cervix, 151 cases of LSIL, and 55 cases of high-grade squamous intraepithelial lesion (HSIL) and above (HSIL(+); including 53 cases of HSIL, 1 case of cervical adenocarcinoma, and 1 case of cervical squamous cell carcinoma). (2) The Ct value of HPV 16 was inversely correlated with the upgrading of the lesions (r=-0.617, P=0.000), and significant different among normal cervix,LSIL and HSIL(+) (35.4±4.5 vs 31.0±6.0 vs 26.5±4.0; F=25.537, P=0.000). There was no correlation between Ct value of HPV 18 and cervical lesions (r=-0.021, P=0.902). The Ct value of other 12 HPV types was statistically difference among normal normal cervix, HSIL(+) and cervicitis (33.0±5.3 vs 29.9±7.2 vs 29.8±5.8; F=5.087, P=0.007). Among them, LSIL and HSIL(+) were significantly lower than normal cervix (P<0.05), but there was no significant difference between LSIL and HSIL(+) (P>0.05). Conclusion: The Ct value of HPV 16 detecting in Cobas 4800 system as an indicator of virus load obviously correlates with different grades of cervical lesions, therefore could be a reference of cervical lesion existence and an indicator of lesion prognosis.

[Value of the BioPerfectus multiplex real time HPV assay for self-collected samples cervical cancer screening].

Objective: To evaluate the feasibility of the BioPerfectus multiplex real time (BMRT) HPV assay for self-sample cervical cancer screening. Methods: Eight hundreds and thirty-nine self-collected and physician-obtained DNA samples from the Shenzhen cervical cancer screening trial Ⅳ(SHENCCAST-Ⅳ) study collected samples for cervical cancer screening during June 2013 to September 2014 were detected by BMRT HPV assay to evaluate the screening efficacy. Results: A total of the 839 women who were screened, 804 with complete BMRT HPV data was included in the study, and average age was (46±7) years. Of the 804 women, the positive rates of 14 high-risk HPV genotypes (including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 subtype) of self-sample and physician-obtained samples were 12.2% (98/804) and 12.8% (103/804), respectively (χ(2)=0.14, P=0.71). Self-collected samples with HPV-positive had significantly more cells (median 19 901.0) than physician-obtained samples (median 1 778.4), and there was statistically significant difference (Z=-7.61, P<0.01). The degree of agreement between self-sample and physician-obtained samples of HPV 16, HPV 18 and other 12 high risk HPV genotype was 99.8%, 100.0% and 96.1%, respectively. And the consistent Kappa value was 0.95, 1.00 and 0.81, respectively. Of 804 samples, there were 6 cervical intraepithelial neoplasia (CIN)Ⅱ(+) cases. There were no missed CINⅡ(+) cases by BMRT HPV assay. Conclusion: BMRT HPV assay is feasible for self-sample cervical cancer screening.

Understanding m6A Function Through Uncovering the Diversity Roles of YTH Domain-Containing Proteins.

N6-methyladenosine (m6A) is the most abundant-internal modification of eukaryotic mRNA. m6A can be installed and removed by specific enzymes. The "writer," "eraser," and "reader" of m6A modification have been reported. These discoveries facilitate our understanding of the functional significance of m6A. m6A plays an essential role in diverse biological processes by recruiting the corresponding YTH domain-containing proteins, as well as recruiting additional translation initiation factors. Here, we provide an update on the various aspects of YTH domain-containing proteins, including an introduction to the YTH domain, the categories, distribution in cells, and biological roles of YTH proteins. Then we focus on the mechanisms that YTH proteins recognize m6A and mediate the fate of methylated-RNAs in eukaryotic cells.

[Characteristics of invisible cervical intraepithelial neoplasia â ¢ under colposcopy].

Objective: To explore the human papilomavirus (HPV) genotypes and epithelial thickness of invisible cervical intraepithelial neoplasia Ⅲ (CIN Ⅲ) under colposcopy. Methods: One hundred and sixty-nine biopsies from 93 patients with a final diagnosis of CIN Ⅲ were extracted from the Shenzhen cervical cancer screening trial Ⅱ (SHENCCAST Ⅱ) . The SHENCCAST Ⅱ was conducted from 2009 to 2010. All the cervical blocks from these patients were re-cut and placed on 6 slides, i.e. sandwich model, with the top and bottom sections being stained with HE, the top second be processed for other studies, 3 sections for HPV genotypes by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) assay. The thickness of squamous epithelium of CIN Ⅲ was measured by a microscope (×10) after re-cut. Colposcope directed CIN Ⅲ biopsies positively was defined as visible CIN Ⅲ, while random CIN Ⅲ biopsies positively was defined as invisible CIN Ⅲ. Results: HPV16 positivity was 37.2% (16/43) and 55.6% (70/126) between invisible and visible CIN Ⅲ biopsies, respectively (χ(2)=4.318, P=0.038) . Forty-nine cases of the 93 CIN Ⅲ patients were HPV16 positive, while 44 of them non-HPV16 positive. The proportion of patients with ≥45 years of age for other non-HPV16 positive 40.9% (18/44) was significantly higher than that HPV16 positive 20.4% (10/49; χ(2)=4.630, P=0.031) . Patients with HPV16 positive were more likely to have lesions ≥1 quadrant (χ(2)=7.786, P=0.005) than other non-HPV16 positive. Compared the average epithelium thickness of invisible CIN Ⅲ tissue (140±12) µm, the average epithelium thickness of visible CIN Ⅲ tissue (161±9) µm was thicker. There was statistical difference between two groups (t=4.383, P=0.038). The mean average epithelial thickness of CIN Ⅲ with HPV16 positive (172±11) µm was thicker than that the mean average epithelial thickness of CIN Ⅲ with non-HPV16 positive (130±10) µm (t=4.784, P=0.031) . Conclusions: Invisible lesions is difficult to identify under colposcopy and is related to non-HPV16 positive, small lesion size and thinner squamous epithelium. For non-HPV16 positive or older women should be performed colposcope directed biopsies and randomly multi-sites biopsies by colopscopy, which may be helpful to improve the detection of CIN Ⅲ and to reduce miss diagnosis.

[Evaluation of CIN2+ /CIN3+ risk of different HPV subtypes infection combined with abnormal cytology status].

Objective: To determine the morbidity of cervical intraepithelial neoplasia 2+ (CIN2+ ) and CIN3+ of different human papillomavirus(HPV) subtype infection combined with different cytology status. Methods: The Shenzhen Cervical Cancer Screening Trial Ⅰ & Ⅱ (SHENCCASTⅠ&Ⅱ) are population-based cross-sectional cervical cancer screening studis conducted in Shenzhen and surrounding area from 2008 to 2010. A total of 12 097 women who aged 25-59 years were included in the analysis. All of these women were detected by liquid-based cytology test and several high-risk HPV-DNA tests. The ones with HPV positive or atypical squamous cells of undetermined sign (ASC-US) were sequentially conducted by cervical biopsy vaginoscopy. Finally, 10 805 samples with complete data of hybrid capture 2(HC2), the polymerase chain reaction-based matrix-assisted laser desorption/ionization time-of-flight assay (MALDI-TOF), HPV genotyping detection, cytology and pathology results were analyzed. Results: The top 6 infection rates of HR-HPV in CIN2+ and CIN3+ were HPV16, HPV52, HPV58, HPV33, HPV31, HPV18. The highest constituent ratio of cytology in CIN2+ and CIN3+ was high grade squamous intraepithelial lesion(HSIL). The morbidities of CIN2+ of patients infected with HPV16, HPV31, HPV58, HPV33, HPV18, HPV52 were 41.3%, 31.5%, 30.6%, 28.7%, 28.2%, 17.7%, respectively, while the morbidities of CIN3+ of those were 33.5%, 20.5%, 19.4%, 15.7%, 19.2%, 8.3%, respectively.The morbidities of CIN2+ in negative intraepithelial lesion or malignancy (NILM), ASC-US, low grade squamous intraepithelial lesion (LSIL), atypical squamous cell cannot exclude high-grade squamous intraepithelial lesion (ASC-H), high grade squamous intraepithelial lesion (HSIL), atypical glandular cell (AGC) samples were 0.4%, 6.9%, 11.1%, 36.4%, 82.0%, 16.7%, respectively, while the morbidities of CIN3+ of those were 0.2%, 3.1%, 4.2%, 22.7%, 64.8%, 0.0%, respectively. The morbidities of CIN2+ in NILM combined with HPV16, HPV18, HPV31, HPV33 infection were 12.6%, 13.3%, 15.8% and 11.5%, respectively, while the morbidities of CIN3+ of those were 10.3%, 11.1%, 7.9% and 7.7%, respectively.The morbidities of CIN2+ and CIN3+ in ASC-US combining with hrHPV infection were high, and the top 6 subtypes associated with high risk of CIN2+ were HPV31 (35.7%), HPV33 (26.9%), HPV16 (26.5%), HPV58 (22.4%), HPV52 (18.6%), HPV68 (15.4%), while those associated with high risk of CIN3+ were HPV16 (20.4%), HPV31 (14.3%), HPV33 (11.5%), HPV58 (8.6%), HPV68 (7.7%), HPV52 (5.8%). Conclusions: Cytology combined with HPV genotyping detection can more effectively estimate the morbidity risks of CIN2+ and CIN3+ . Both high prevalence rates and high risks associated with CIN2+ and CIN3+ of HPV31, HPV33, HPV52 and HPV58 are observed. NILM and ASC-US status combined with these subtypes mentioned above are advised to be conducted by colposcopy.

In vivo osseointegration of dental implants with an antimicrobial peptide coating.

This study aimed to evaluate the in vivo osseointegration of implants with hydrophobic antimicrobial GL13K-peptide coating in rabbit femoral condyles by micro-CT and histological analysis. Six male Japanese Rabbits (4 months old and weighing 2.5 kg each) were included in this study. Twelve implants (3.75 mm wide, 7 mm long) were randomly distributed in two groups, with six implants in the experimental group coated with GL13K peptide and six implants in the control group without surface coating. Each implant in the test and the control group was randomly implanted in the left or right side of femoral condyles. On one side randomly-selected of the femur, each rabbit received a drill that was left without implant as control for the natural healing of bone. After 3 weeks of healing radiographic evaluation of the implant sites was taken. After 6 weeks of healing, rabbits were sacrificed for evaluation of the short-term osseointegration of the dental implants using digital radiography, micro-CT and histology analysis. To perform evaluation of osseointegration, implant location and group was double blinded for surgeon and histology/radiology researcher. Two rabbits died of wound infection in sites with non-coated implants 2 weeks after surgery. Thus, at least four rabbits per group survived after 6 weeks of healing. The wounds healed without suppuration and inflammation. No implant was loose after 6 weeks of healing. Radiography observations showed good osseointegration after 3 and 6 weeks postoperatively, which proved that the tissues followed a natural healing process. Micro-CT reconstruction and analysis showed that there was no statistically significant difference (P > 0.05) in volume of bone around the implant between implants coated with GL13K peptide and implants without coating. Histomorphometric analysis also showed that the mineralized bone area was no statistically different (P > 0.05) between implants coated with GL13K peptide and implants without coating. This study demonstrates that titanium dental implants with an antimicrobial GL13K coating enables in vivo implant osseointegration at similar bone growth rates than gold-standard non-coated dental implants up to 6 weeks of implantation in rabbit femurs.

Evaluation of novel assays for the detection of human papilloma virus in self-collected samples for cervical cancer screening.

The aim of this study was to evaluate the performance of three new high-risk human papillomavirus (HPV) assays for primary cervical cancer screening, by using self-collected samples, and to identify an HPV assay that could overcome the major obstacles faced during large-scale population-based screening. Two hundred and ten women showing abnormal cervical cytology (and referred for a colposcopy) were recruited in this study. Self-collected samples obtained from all women were tested with the Cobas, Seq, and BioPerfectus Multiplex Real Time HPV assays; simultaneously, clinician-collected samples (from the same women) were tested with the gold-standard Cobas HPV assay. The results of all the assays were consistent. The sensitivity, positive predictive value, and negative predictive value for cervical intraepithelial neoplasia 2+ (CIN2+) and CIN3+ were comparable between the self-collected samples tested with the three new assays and the clinician-collected samples tested with the Cobas HPV assay (P > 0.05). The single-genotype HPV load per sample did not differ significantly between the self- and clinician-collected samples (P = 0.195). In conclusion, the results of this study demonstrated the applicability of the three new HPV assays for primary cervical cancer screening based on self-collection.

p66(Shc) restrains Ras hyperactivation and suppresses metastatic behavior.

Normal tissue cells survive and proliferate only while anchored to solid substrate. Conversely, transformed cells both survive and proliferate following detachment, having lost attachment context through unclear mechanisms. p66(Shc) is a focal adhesion-associated protein that reports cell attachment through a RhoA-dependent mechanosensory test. We find that human small cell lung cancer (SCLC) cells and mouse Lewis lung carcinoma (LLC), which display aggressive metastatic behavior, lack both p66(Shc) and retinoblastoma (pRB) and bypass anoikis. Re-expression of p66(Shc) in these cells restores anoikis and provides striking protection from metastasis by LLC cells in vivo. Notably, knockdown of p66(Shc) in normal epithelial cells leads to unrestrained Ras activation, preventing anoikis through downstream suppression of RhoA but blocking proliferation in a pRB-dependent manner, thus mimicking oncogenic Ras. Conversely, LLC and SCLC cells display constitutive Ras activation necessary to bypass anoikis, which is reversed by re-expression of p66(Shc). p66(Shc) therefore coordinates Ras-dependent control of proliferation and anchorage sensation, which can be defeated in the evolution of highly metastatic tumors by combined loss of both p66(Shc) and pRB.

Different cervical cancer screening approaches in a Chinese multicentre study.

To evaluate alternative cervical cancer screening methods, digital colposcopy and collection of cervical exfoliated cells for liquid-based cytology (LBC) and hybrid capture 2 (HC2) testing were performed among 2562 women aged 15-59 years in three study sites in the People's Republic of China (rural Shanxi province, Shenyang city in Liaoning province and Shenzhen city in Guangdong province). Visual inspection with acetic acid (VIA) was also evaluated independently from colposcopy. A total of 74 cases of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) were identified, and 16 CIN2+ cases were imputed among unbiopsied women to correct for verification bias. Corrected sensitivity for CIN2+ was 37% for VIA, 54% for colposcopy, 87% for LBC with a threshold of atypical cells of undetermined significance (LBC>or=ASCUS), 90% for HC2, 84% for LBC using HC2 to triage ASCUS and 96% for positivity to LBC>or=ASCUS or HC2. For VIA, sensitivity was much lower among women >or=40 years (12%) than those aged or=ASCUS or HC2, up to 94% for LBC using HC2 to triage ASCUS. In conclusion, LBC, HC2 and their combinations performed well, whereas VIA missed a majority of CIN2+, particularly in older women. Digital colposcopy performed better than VIA, but still missed nearly half of CIN2+ in this study.

HIV Tat activates c-Jun amino-terminal kinase through an oxidant-dependent mechanism.

The HIV-1 accessory protein Tat has been found to exert profound effects on vascular cell behavior. Recently, Tat has been found to activate the c-Jun amino-terminal kinase (JNK1, SAPK) MAP kinase in lymphoid cells. We found that purified Tat rapidly activated JNK1 in human umbilical vein endothelial cells and ECV-304 cells, and coculture of ECV-304 cells with Tat-transfected HeLa cells resulted in persistent activation of JNK1. In addition, lower doses of Tat potentiated TNFalpha-induced JNK1 activation, although higher doses paradoxically diminished JNK1 activation by TNFalpha. Treatment of ECV-304 cells with Tat acutely increased intracellular oxidant levels, and Tat-induced oxidant activity was decreased by two structurally distinct NADPH oxidase inhibitors, diphenylene iodonium and apocynin. Both oxidase inhibitors and the thiol antioxidant N-acetyl cysteine decreased Tat-induced JNK1 activation in parallel with reduction in oxidant levels. Activation of JNK1 by Tat was also inhibited by cytochalasin B, suggesting that Tat signaling was dependent upon intact cytoskeletal function. Indeed, JNK1 activation by Tat was associated with actin microfilament rearrangement. We conclude that HIV Tat may cause acute and persistent activation of the JNK MAP kinase through activation of a specific oxidase.

A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid synthase genes.

Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.